The present invention relates to a flow regulator apparatus, and a system employing the same, which regulates the flow of fluid through the system to maintain the flow of fluid at a controllable volumetric flow rate. More particularly, the present invention relates to a flow regulator that is employed in, for example, a sheath fluid delivery system of a flow cytometer, to regulate the flow of sheath fluid so that the fluid flows at a constant volumetric flow rate which is unaffected by the pressure, temperature, and viscosity of the fluid.
Many different types of devices for regulating the flow of fluid through a fluid delivery system are known in the art. For example, as described in U.S. Pat. No. 1,887,322, a valve is employed in a train brake pipe that is supplied with fluid under pressure to regulate the pressure of the fluid in the brake pipe. U.S. Pat. No. 2,742,764, on the other hand, describes a system for supplying liquid anhydrous ammonia to soil as a fertilizer, in which is employed a device for regulating the flow of the liquefied anhydrous ammonia through the system. Flow regulators are also employed in exhaust systems in automobiles, as described in U.S. Pat. No. 3,520,312, and are further employed in gas mixing systems as described in U.S. Pat. No. 3,392,752.
Certain types of medical devices include fluid delivery or distribution systems which employ devices that regulate the flow of fluid to the system. For example, in an infusion pump or IV pump, or in a dialysis system, it is necessary to regulate the flow of blood or fluid to the patient. In the art of flow cytometry, it is necessary to regulate the flow of sheath fluid through the sheath fluid delivery system of the flow cytometer.
A flow cytometer is an apparatus for analyzing, and optionally sorting, biological cells or any other particles of interest in a moving fluid stream. Known flow cytometers are described, for example, in U.S. Pat. Nos. 4,347,395, 5,395,588, 5,464,581, 5,483,469, 5,602,039 and 5,643,796, the entire contents of which are incorporated by reference herein. Other known flow cytometers are the FACS Vantage.TM., FACSort.TM., FACSCalibur.TM., FACSCount.TM. and FACScan.TM. systems, all of which are manufactured by Becton Dickinson and Company, the assignee of the present invention.
Flow cytometers such as those described above typically include a sample reservoir for receiving a biological sample, such as a blood sample, which contains cells that are to be analyzed. The cells are transported in a cell stream to an area of the flow cytometer known as a flow cell. A sheath fluid is also directed to the flow cell.
Within the flow cell, the sheath fluid forms a liquid sheath around the cell stream, and the combined sheath fluid and cell stream are focused through a cell analysis region. At the cell analysis region, the cells intercept a laser beam, which causes the laser light to scatter. Furthermore, the laser light excites reagents that have been added to the biological sample, and causes those reagents to fluoresce. An optical detection system, such as a set of photomultiplier tubes, photodiodes or other devices for measuring light, are focused onto the point at which the cells intersect the laser beam (i.e., the interrogation region) to detect the laser light scattered by the cell, fluorescent light emitted from the reagent adhering to the cell, and a portion of the laser beam which passes through the cell. The flow cytometer interprets the light detected by these optical detecting devices to ascertain chemical and physical characteristics of the cell, such as size, granulation of the cytoplasm, and the presence of specific antigens. The flow cytometer can thus count the number of cells in the sample having any of these specific properties.
After passing through the interrogation region, the cell stream is directed through an orifice in the flow cell which forms droplets of sheath fluid, with each droplet containing a respective cell. That is, after each cell passes the interrogation region, it passes through the orifice and becomes suspended in a droplet of sheath fluid. At the moment the droplet is being formed, it is charged with a potential having a magnitude representative of characteristics of the cell being suspended in that droplet. The flow cytometer then sorts these charged droplets, and hence the cells suspended therein, electrostatically or by any other suitable method.
Maintaining a stable sheath fluid flow rate in a flow cytometer is critical for several reasons. For example, some flow cytometers use multiple lasers which emit beams through different locations in the interrogation region in the flow cell. The sheath flow, which governs the movement of the cells through the flow cell, must be stable to insure that the cells intersect the beams at predictable times, so that the scattered light, pass-through light and fluorescent light for each laser beam can be detected at the appropriate time for each cell. In flow cytometers which perform cell sorting, it is necessary to maintain a stable sheath flow rate to insure that the droplet formation for each cell which is to be sorted occurs at a given time after the cell has passed the interrogation region, so that the droplets can be charged based on the detected characteristics of the cell suspended therein.
A known apparatus for directing sheath fluid at a constant flow rate through a sheath fluid delivery system employs a syringe pump. The syringe pump is loaded with a required amount of sheath fluid, and the piston of the syringe pump is advanced at a steady and controlled rate to introduce the sheath fluid into the sheath fluid delivery system at a steady and controlled rate.
Although this apparatus is generally effective in delivering the sheath fluid at a controlled flow rate, the amount of sheath fluid that can be delivered to the system is limited by the size of the syringe pump. Therefore, it is necessary to refill the syringe pump quite often, which is undesirable for tests which require lengthy analysis periods. Furthermore, reliable syringe pumps of these types are expensive and require periodic maintenance. Dual chamber piston pumps, such as the type commonly used in chromatography instrumentation, can overcome the problem of limited fluid volume by delivering the sheath fluid at a constant flow rate through the coordinated action of both pumps. However, dual chamber pumps are more complex and thus, are more costly and require further maintenance than typical single chamber syringe pumps.
Other known systems regulate the sheath fluid flow rate through the use of a vacuum pump, for example, as described in U.S. Pat. No. 5,395,588 to North, Jr., et al., the entire contents of which is incorporated herein by reference. The vacuum pump creates a vacuum downstream of the flow cell to cause the sheath fluid to flow through the fluid delivery system at a steady flow rate. Although this method is effective in providing an essentially uninterrupted flow of sheath fluid, the flow rate of the sheath fluid can change due to a change in temperature of the sheath fluid, as well as due to changes in the liquid level in the source container, and partial clogging of the sheath supply filter. That is, a change in temperature of the sheath fluid will change the viscosity of the sheath fluid, and thus effect the sheath fluid flow rate. Also, since vacuum pumps do not create an unvarying pressure due to mechanical tolerances and so on, such fluctuations in pressure can greatly affect the flow rate of the sheath fluid.
Another known method for regulating flow of fluid includes the use of a flow regulator as described in U.S. Pat. No. 3,749,113. The flow regulator described in this patent includes two flexible diaphragms which create three pressure chambers in the device. The diaphragms are coupled to each other by a member which is coupled to the housing of the device by a spring. Variations in pressure in the pressure chambers cause the diaphragms to move in opposition to the force exerted on the member by the spring until the pressures in the chambers equalize. This pressure equalization maintains a virtually constant flow rate of the fluid flowing through the system.
However, the flexible diagrams employed in this type of regulator are costly, subject to tearing, and make the regulator difficult and costly to manufacture. Furthermore, in order for this type of regulator to operate properly, it must be immersed in a temperature controlled bath. The temperature controlled bath is necessary to maintain constant the temperature of the fluid, and thus maintain constant the viscosity of the fluid passing through the regulator. Additionally, this type of regulator requires the application of pressurized air to the fluid storage chamber, and to one of the chambers in the flow regulator, which further complicates the system.
Accordingly, a continuing need exists for a less complicated flow regulator which is capable of maintaining a constant volumetric flow rate of a fluid through a fluid delivery system, and which is unaffected by changes in fluid temperature, fluid pressure and fluid viscosity.